Figure 4.

The size of Munc18-1 but not Munc18-1^Δ317-333 nanodomains is reduced by secretagogue stimulation. Transfected DKD-PC12 cells expressing Munc18-1^WT-mEos2 (A–C) or Munc18-1^Δ317-333-mEos2 (D–F) were incubated with or without 2 mM BaCl₂ for 2 min in buffer A, then fixed in paraformaldehyde before single-molecule localization (SML) imaging and processing. (A and D) Representative low-resolution (green fluorescence TIRF) image and corresponding SML image of unstimulated (top) and stimulated (bottom) Munc18-1^WT-mEos2 (A) or Munc18-1^Δ317-333-mEos2 (D). Bars: (low-resolution) 2 µm; (enlarged) 200 nm. (B and E) Mean (± SEM) of autocorrelation functions for each region of interest. Three regions of interest were analyzed for each cell, and 10 cells were analyzed for each condition. The experiment was performed three times independently. (C and F) The mean radius (Eq. 1) and the number of molecules per nanodomain (Eq. 2) were obtained from each region of interest by fitting the autocorrelation values. **, P < 0.01, Student’s t test.